Unlike what occurs with individual cells, the study of embryos and similar biological samples through an optical microscope presents particular problems relating to light absorption and resolution loss due to light scattering. To solve these problems, considerable improvements to plane laser beam microscopes, invented in 1903, have been developed in recent years. See for example the document titled “Ultramicroscopy” by Siedentopf and Zsigmondy (Analen der Physik 10(1), 1903). After some minor improvements proposed by Voie et al., in J. Micros. 170, 1993 (technique referred to as OPFOS by the authors), or by Fuchs et al. in Opt. Exp. 2002 referred to as “Thin-sheet imaging microscopy” or TSLIM, in 2004 Stelzer's group presented a plane laser beam microscope referred to as SPIM (Selective Plane Illumination Microscope), having applications in both the image in vivo and in fixed tissue and transparent or semi-transparent samples in general.
A plane laser beam microscope is fundamentally formed by a camera coupled to a objective having a high numerical aperture and arranged according to a direction referred to as “detection direction,” and a lighting means capable of emitting a thin sheet of light according to a direction referred to as “illumination direction” which is perpendicular to the detection direction, following the original configuration by Siedentopf and Zsigmondy coupled to a detection camera. With this configuration, the camera can produce a 2D fluorescence image of the part of the sample illuminated by the sheet or plane of light. If the sample is further moved in the direction of the axis of detection and several 2D images are taken at different positions, a set or stack of 2D images is generated where each of the 2D images corresponds to a position of the plane of light with respect to the sample. This stack of 2D images contains information about the position in z (depth of the sample according to the detection direction) produced by moving the sample, and about positions x and y, present in each 2D image. The stack of 2D images can then be fused together for generating a 3D image of the sample, as described in patent document U.S. Pat. No. 7,554,725 by Stelzer et al.
A drawback of the plane laser beam microscopy technique is that it has worse resolution on the axis of detection than on the plane of the image. In other words, resolution along the x and y axes in the 3D image is more precise than the resolution along the z axis. The multi-view SPIM or mSPIM technique has been developed to solve this anisotropy (see document US 2011/115895 by Huisken). This technique fundamentally consists of including an additional illumination arm for producing at least two illumination measurements opposite one another by 180°. Image resolution can be enhanced by means of a slight pivoting of less than 10° of the light plane on the plane of illumination. If an additional camera is also included, four simultaneous measurements corresponding to all the possible combinations between camera/illumination arm can be taken. These 2D images are later fused together for generating a single higher quality 3D image of the sample in question.
Another one of the proposed ways to enhance image anisotropy and quality is to combine several angular measurements in a single 3D measurement. In other words, the sample is rotated about its own axis, usually a vertical axis, such that several stacks of 2D images are captured (commonly referred to as “angular measurements”), each of which corresponds to a different angle of rotation of the sample. This was the proposal published by S. Preibisch et al., Nature Methods 7 (2010), who propose the use of reference fiducials in order to suitably align these angular measurements.
To better understand this technique, FIGS. 1a and 1b are enclosed, showing two examples of plane laser beam microscopes (100). In FIG. 1a, the sample (107) is arranged on a support (101) in a bath (102). A Gaussian, Bessel or Airy linear light beam (103) strikes a cylindrical lens (104) that focuses it as a result of an illumination objective (105) for generating the vertical plane light sheet (106). This vertical plane light sheet (106) strikes the sample (107) according to the illumination direction (DI), and the fluorescent light (108) is picked up by a detection objective (109) oriented according to the detection direction (DD), which is perpendicular to the illumination direction (DI). FIG. 1b shows a similar microscope (100), although in this case the formation of the plane light sheet (106) takes place by means of the vertical scanning of the linear light beam (103) by means of a galvanometric mirror (104′) or the like. In both cases, the support (101) can rotate about its vertical axis in order to allow taking several angular measurements according to the technique proposed by Preibisch.
FIG. 2 shows a detail of the formation of a stack of 2D images of the sample with a plane laser beam microscope (100) according to FIG. 1a or 1b. It can be seen how the sheet (106) moves according to the detection direction, one 2D image being taken for each of said positions. The final result is to produce a stack of 2D images. For carrying out the method proposed by Preibisch et al., this process is repeated several times for different angles of rotation of the sample about the vertical axis, which allows producing a 3D image of the sample with greater isotropy.
However, the introduction of these angular measurements entails increasing exposure time and the duration of the experiment in a manner that is proportional to the number of angular measurements. Indeed, given that the exact position exacta of the center of rotation is unknown, fusing all the angular images that are produced in a plane laser beam microscope requires the use of fiducials for generating the final 3D image (see S. Preibisch, et al., Nature Methods 7, 2010), which requires enormous computing power and storage capacity and complicates experimental measurement.